Blood coagulation tests are conducted on plasma samples from humans/animals by making blood samples incoagulable with sodium citrate (volume ratio blood/anticoagulant 9:1). To conduct the determination of individual measurement values of the blood coagulation, blood is centrifuged and the cells (in the sediment) are separated from the liquid blood components (plasma). To measure the blood coagulation, calcium chloride and an activator of the blood coagulation are added to the plasma. In special methods for determining individual clotting factors or exogenous inhibitors, photometric proof is used. In these methods, the colorant para-nitroaniline is released by an exogenously added coagulation enzyme (e.g. factor Xa) from a chromogenic substrate (N-benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine-para-nitroaniline hydrochloride (SEQ ID NO:01) and others), the activity/concentration of factor Xa inhibitors or substances inhibiting factor Xa being measured in a concentration-dependent manner. The result is a linear or sigmoidal decrease of the release of para-nitroaniline, measured at 405 nanometers in the photometer, depending on the concentration of the factor Xa inhibitor (Harenberg J, Modified anti-factor Xa chromogenic substrate assay for heparin and low molecular weight heparins Arztl. Lab. 1987, 33: 39-41).
For other tests in blood, such as liver enzymes, kidney parameters, electrolytes or cholesterol, blood is coagulated by the addition of an activator (e.g. kaolin). Here, the coagulation proteins, antithrombin and fibrinogen are used. These factors are mixed with blood cells in a blood clot in the coagulum in a coagulation tube, into which blood is withdrawn. Serum can be found in the supernatant, which does not contain these coagulation proteins. The measurement of these clinical-chemical parameters is easier in serum. Concentrations of drugs are measured in serum as well, except for the coagulation drugs for clinical use.
Heparins, low-molecular-weight heparins, heparinoids, fondaparinux and other polysaccharides need cofactors in the blood (antithrombin, heparin cofactor II) to activate their anticoagulative effect or to accelerate it by as much as 1000 times. These cofactors are present in plasma, so that citrate plasma samples are used for analyzing the activity of the inhibitors of blood coagulation. Tests in serum are not possible with methods for the clinical routine (Harenberg J, Neue Antikoagulantien. Zett Verlag, Steinen, 2007).
Other inhibitors of blood coagulation do not need cofactors in the blood to become active. These are so-called direct coagulation inhibitors of coagulation enzymes. The most important ones for the time being are the group of direct factor Xa and thrombin inhibitors. Their activity/concentration is measured in citrate plasma with different methods/activators. Rivaroxaban is the first one of the oral direct factor Xa inhibitors to be clinically used. The relevant determination methods for detecting the concentration/activity of rivaroxaban are published. All analyses are made with plasma anticoagulated with citrate (Samama M M, Martinoli J, Leflem L, et al. Assessment of laboratory assays to measure rivaroxababan—on oral, direct factor Xa inhibitor. Thromb Haemost 2010; 103: 815-825; Tripodi A, Guinet C, Samama M. The Internationalized Normalized Ratio (INR) Calibrated for Rivaroxaban (INRivaroxaban) Normalizes Prothrombin Time Results for Patients Treated with this Drug. J Thromb Haemost 2010, online available; Harenberg J, Marx S, Kramer R et al. Reduction of variability between prothrombin time reagents of plasma samples containing rivaroxaban using the WHO/RBT 90 thromboplastin reagent, submitted for publication).
So far, there have not been any detection methods in the clinical-chemical routine for inhibitors of blood coagulation from serum. Serum has different advantages over plasma: In medicine, serum samples are more often taken from patients than plasma samples. The blood withdrawal is less susceptible to influences for serum samples than for plasma samples. In the case of a “bad” blood withdrawal, blood coagulation can be activated. Thereby, the results are influenced by the clotting factors. This is not possible for serum samples, since a blood coagulation is performed in the tube after the withdrawal.
Current solutions have the disadvantage of requiring a separate blood withdrawal for obtaining plasma samples for the analysis. Antithrombin, factor X/Xa and other coagulation proteins are contained in the patients' plasma in different quantities. This influences the test result. A blood withdrawal involves the risk of local side effects, such as hematoma, or generalized side effects, such as inflammation of the vein or transmission of infection (e.g. hepatitis, HIV).
Thus, the problem underlying the present invention is to provide new means for an efficient detection of factor Xa inhibitors which overcome the shortcomings of the protocols known in the art.